Ported parallel plate flow chamber and methods for use thereof

ABSTRACT

Flow chambers are provided. In some embodiments, the flow chambers include an inner panel having at least one flow channel having an inlet/outlet opening on each end thereof formed therein, wherein the inlet/outlet openings are adapted to releasably receive a septum; one or more ports adapted to releasably receive a plug and for at least liquid communication with the at least one flow channel, and an outer frame that defines an outer portion of the at least one flow channel and that defines a perimeter of the flow chamber. In some embodiments, the flow chamber has overall dimensions of a standard multiwell plate and the at least one flow channel is located in a position that corresponds to a column location of the standard multiwell plate. Also provided are methods for producing the presently disclosed flow chambers and employing the same to assay biological features of cultured cells and/or tissues.

CROSS REFERENCE TO RELATED APPLICATION

The presently disclosed subject matter claims the benefit of U.S.Provisional Patent Application Ser. No. 61/791,770, filed Mar. 15, 2013,the disclosure of which is incorporated herein by reference in itsentirety.

TECHNICAL FIELD

The presently disclosed subject matter relates generally to parallelplate flow chambers and methods for using the same to examine theeffects of different fluid flows on cells and biological activitiesthereof. In particular, the presently disclosed subject matter relatesto apparatuses on which cells and/or tissues can be cultured and testedfor responses to different fluid flow environments.

BACKGROUND

In vitro cell culture is routinely performed as part of a wide varietyof biological research and development programs. In their most commonform, cell based experiments are carried out in culture dishes or flasksunder static conditions—i.e., those in which no external forces areapplied to the cells. Work conducted with statically grown cells has ledto many breakthroughs in fields such as cell biology, biochemistry,immunology, and cancer research. However, the inability of staticculture to accurately mimic the behavior of cells in dynamic tissueenvironments constitutes a boundary on the usefulness of this technique.This is illustrated by the number of drug candidates that fail at thetransition from in vitro to in vivo testing. It is well known thatenvironmental forces (such as those derived from the flow of blood andother interstitial fluids) influence the behavior of cells and tissuesin determining states of health and disease and responses tobiochemicals (Buchanan et al., 1999; Urbich et al., 2001; Wasserman &Topper, 2004; Sheikh et al., 2005; Chatzizisis et al., 2007; Chiu etal., 2007; Tsai et al., 2007). Similarly, these forces can also modulatecellular responses to pharmaceuticals, and influence their ultimateefficacy profiles. Since static culture is incapable of introducingvariables such as fluid flow into experimental design, alternative meansof cell cultivation are necessary to investigate the influence ofphysiological forces on cell behavior, both in native environments andin response to biochemicals and pharmaceuticals.

The effects of blood flow on cell physiology were first observed in thecontext of arterial cells susceptible to developing arterial (heart)disease, but other physiological phenomena, such as immune cellrecruitment, wound-healing, stem cell differentiation, and tissueregeneration are also known to be force dependent (Rinker et al., 2001;Dekker et al., 2002; Burns & DePaola, 2005; LaMack et al., 2005;Yamamoto et al., 2005; McKinney et al., 2006). Due to the prevalence ofheart disease in western society, the effect of fluid flow has become aprimary topic of investigation for those interested in understanding itspathology and developing novel treatments. Due to the dependence of thedevelopment and progression of heart disease on the characteristics ofarterial blood flow, much research is focused upon understanding howvarious fluid forces influence cell physiology. This work cannot beperformed under static conditions, but instead requires the use ofdynamic culture systems. Similarly, investigations into the other forcedependent physiological processes mentioned above have related culturesystem requirements. Unfortunately, there has been no commerciallyavailable consumable device flexible enough to support the variety offluid force based cell culture research and development that is beingconducted. Instead, most academic and commercial laboratories havecreated their own systems, while a large number of other entities thatwould like to perform such experiments do not, as they consider the needto fabricate and assemble the required apparatus as a significantbarrier to practice.

In addition to the areas of research and development currentlyinvestigated in flow systems, there is a need to expand this approach tothe drug discovery pipeline. The same blood vessel cells involved inheart disease serve as gatekeepers for drugs entering the bloodstream,and participate in determining their efficacy (McNeish, 2004; LaMack etal., 2005). Kidney tubular epithelial cells and liver sinusoidalepithelial cells are involved in drug metabolism and excretion, aresubject to fluid flow, and their flow sensitivity has been reported(Duan et al., 2010; Essig and Friedlander, 2003; Shah et al., 1997). Byconducting initial screening experiments and later toxicity/therapeuticstudies with cell cultures exposed to conditions similar to those thatexist within the body, results will be more closely linked to actualbehavior in tissue, and the economics of the process improved. It is ourbelief that this can only be achieved through the use of a device suchas the chamber device proposed in this application. These outcomes willallow pharmaceutical companies to identify high value candidatesearlier, to understand their properties more completely, and to focustheir resources on only those molecules that meet the more realistic setof physiological criteria.

There are two common types of devices that support cell and tissueexperiments in a dynamic fluid environment. The first of these is theparallel plate flow chamber. Parallel plate flow chambers consist of twoparallel plates separated by a gap that forms the flow channel. This gapis generally created by a gasket or spacer that is used tosimultaneously seal the flow channel and separate the plates. Fluid isintroduced from one end of the chamber and exits on the one opposite.Parallel plate devices are commonly used for exposing cells to definedlevels of shear stress, applying specific flow characteristics, and forinvestigating cell to cell or cell to substrate attachment properties(Frangos et al., 1985; Rinker et al., 2001; McKinney et al., 2006;Shepherd et al., 2009; Shepherd et al., 2011). The other type of deviceconsists of a cone and plate viscometer that has been modified tosupport cell cultures. In these systems, cells may be exposed to variouslevels of fluid shear stress and flow waveforms created by the rotationof the cone (Dai et al., 2004). Neither of these systems are currentlycommercially available for large scale culture activities. Some flowchambers based on a parallel plate design are being marketed bycompanies such as Ibidi, Fluxion, Cellix, Cellasics, IntegratedBiodiagnostics, and Glycotech; however most are based upon smallmicrofluidic flow channels, and do not provide for a wide variety offlow conditions or readout modalities. Additionally, some chambers haveissues generating uniform flow (and hence shear stress) distribution(Nauman et al., 1999; Brown & Larson, 2001; McCann et al., 2005;Anderson et al., 2006).

Described herein are flow chambers with differing geometries, obstacles,gap widths, wall heights, etc. designed to provide finely tunable flowconditions, as well as methods of making and using the same to assayvarious biological properties of cells and/or tissues experiencingdifferent flow conditions.

SUMMARY

This Summary lists several embodiments of the presently disclosedsubject matter, and in many cases lists variations and permutations ofthese embodiments. This Summary is merely exemplary of the numerous andvaried embodiments. Mention of one or more representative features of agiven embodiment is likewise exemplary. Such an embodiment can typicallyexist with or without the feature(s) mentioned; likewise, those featurescan be applied to other embodiments of the presently disclosed subjectmatter, whether listed in this Summary or not. To avoid excessiverepetition, this Summary does not list or suggest all possiblecombinations of such features.

The presently disclosed subject matter provides in some embodiments aflow chamber. In some embodiments, the flow chamber comprises (a) aninner panel having at least one flow channel formed therein, wherein theat least one flow channel has an inlet/outlet opening on each endthereof, and further wherein the inlet/outlet openings are adapted toreleasably receive a septum; (b) one or more ports adapted for at leastliquid communication with the at least one flow channel to permit liquidor and/or a reagent to be added the at least one flow channel, saidports adapted to releasably receive a plug, and optionally wherein theone or more ports are adapted to provide a liquid-proof seal to the atleast one flow channel, and further optionally wherein the ports areadapted to be resealable; and (c) an outer frame that defines an outerportion of the at least one flow channel and that defines a perimeter ofthe flow chamber. In some embodiments, the inlet/outlet openingscomprise a recess adapted to receive the septum. In some embodiments,the outer frame comprises a surface upon which cells can be grown inculture. In some embodiments, the presently disclosed flow chambercomprises two or more flow channels, optionally three, four, five, six,seven, eight, nine, ten, eleven, twelve, or more flow channels.

In some embodiments, the flow chamber has overall dimensions of astandard 6, 12, 24, 48, 96, 384, or 1024 well multiwell plate and the atleast one flow channel is located in a position that corresponds to acolumn location of a standard 6, 12, 24, 48, 96, 384, or 1024 wellmultiwell plate. In some embodiments, the flow chamber has the overalldimensions of a standard multiwell plate such as a standard 96 well or384 well multiwell plate, and each of a series of virtual wells ispresent in a location aligned with a well position of a standardmultiwell plate such as a standard 96 well or 384 well multiwell plate.In some embodiments, the presently disclosed flow chamber comprises two,three, four, five, six, seven, eight, or up to 12 flow channels, each ofwhich is individually located in a column position that corresponds to adifferent column is location of a standard 96 well plate. In someembodiments, the overall dimensions of the flow chamber are consistentwith ANSI/SBS multiwell plate standards such as ANSI/SBS 96 or 384 wellmultiwell plate standards.

In some embodiments, the at least one flow channel has dimensions ofbetween about 5 and 80 mm long by about 1 and 20 mm wide by about 0.025and 2.5 mm high. In some embodiments, the at least one flow channel ischaracterized by one or more gaps, obstacles, and/or other modificationsdesigned to create one or more variable fluid dynamic conditions withinthe at least one flow channel. In some embodiments, the at least oneflow channel has an increasing flow channel height along at least aportion of its length. In some embodiments, the flow channel heightincreases in a plurality of steps.

In some embodiments, at least an inner surface of the at least one flowchannel is chemically and/or physically treated and/or is functionalizedby reactive groups and/or by macromolecules.

In some embodiments, the presently disclosed flow chamber comprises aseptum adapted for placement in one of the inlet/outlet openings. Insome embodiments, the septum is adapted to be liquid tight when thefirst inlet/outlet opening, the second inlet/outlet opening, or both arein fluid communication with the at least one flow channel. In someembodiments, each inlet opening, each outlet opening, or allinlet/outlet openings comprise a septum placed therein.

In some embodiments, at least one of the one or more ports comprisesfitted therein a polymer plug, optionally a gas permeable plug. In someembodiments, the one or more ports comprise one or more hydrophobicpolymer plugs, optionally one or more hydrophobic porous or non-porouspolymer plugs. In some embodiments, the one or more hydrophobic polymerplugs are self-sealing, optionally self-sealing within a port. In someembodiments, the one or more ports comprise one or more plugs adapted toaccept a standard pipettor shaft, a standard micropipettor shaft, anautomated liquid handler head or tip, or any combination thereof. Insome embodiments, the one or more ports comprise one or more plugs thatare hollow. In some embodiments, the one or more plugs are adapted forconnection to one or more gas filters, optionally wherein the one ormore gas filters has a porosity of at most 0.2 μm.

In some embodiments, the inner panel, the outer frame, or both compriseone or more view windows through which the at least one flow channel ora cell growing thereupon can be observed. In some embodiments, thepresently disclosed flow chamber further comprises one or more viewingwindows positioned within the perimeter defined by the skirt and betweenthe welding ribs. In some embodiments, the one or more viewing windowsare located above or below a flow channel, optionally over or under theentire length of a flow channel. In some embodiments, the one or moreviewing windows are characterized by a thinner wall in the outer frameor inner panel than is present in the outer frame or inner panel atpositions other than directly under or over the flow channel. In someembodiments, the inner panel, the outer frame, the one or more viewwindows, or any combination thereof are made from one or more plasticsthat are non-birefringent, non-auto-fluorescent, or both. In someembodiments, the outer frame comprises bottom viewing windows that aremade of glass.

In some embodiments, the outer frame comprises a skirt defining aperimeter and welding ribs positioned along the bottom of the flowchamber. In some embodiments, the outer frame (i) is adapted to seal theseptum in its corresponding inlet/outlet opening; and/or (ii) comprisesone or more holes to access the septum for fluidics connections.

In some embodiments, the flow chamber is adapted for sealing byultrasonic welding of the inner panel and the outer frame.

In some embodiments, the inner panel and the outer frame are produced byinjection molding.

In some embodiments, the flow chamber of the presently disclosed subjectmatter is provided as a preassembled, presterilized, liquid tight, andtissue culture ready device.

In some embodiments, the flow chamber of the presently disclosed subjectmatter further comprises at least a first liquid reservoir that is influid communication with the at least one flow channel via a first lineattached to the first inlet/outlet opening. In some embodiments, thefirst liquid reservoir is contained within the flow chamber device.

The presently disclosed subject matter also provides a flow chambercomprising (a) an inner panel having at least one flow channel formedtherein, wherein the at least one flow channel has an inlet/outletopening on each end thereof, and further wherein the inlet/outletopenings are adapted to releasably receive a septum; (b) one or moreports adapted for at least liquid communication with the at least oneflow channel to permit liquid or and/or a reagent to be added the atleast one flow channel; and (c) an outer frame that defines an outerportion of the at least one flow channel and that defines a perimeter ofthe flow chamber; wherein (i) the outer frame has a footprint equivalentto that of a standard multiwell plate such as a standard 96 well or 384well multiwell plate; (ii) each of the at least one flow channels islocated in a position that corresponds to a column location of astandard multiwell plate such as a standard 96 well or 384 wellmultiwell plate; and (iii) each of the at least one flow channelscomprises a plurality of virtual wells, each virtual well is located ina position that corresponds to a well location of a standard multiwellplate such as a standard 96 well or 384 well multiwell plate. In someembodiments, the presently disclosed flow chamber further comprises oneor more contact points adapted to facilitate interaction of the flowchamber with an automated plate handling apparatus, a multiwell platereader, an automated microscopy system or any combination thereof. Insome embodiments, the inner panel comprises a surface upon which cellscan be grown in culture. In some embodiments, the flow chamber of thepresently disclosed subject matter comprises one, two, three, four, six,or twelve flow channels.

The presently disclosed subject matter also provides methods forproducing the presently disclosed flow chambers. In some embodiments,the methods comprise assembling the inner panel and the outer frame ofany embodiment of the presently disclosed flow chambers andultrasonically welding the inner panel to the outer frame, optionallyvia welding ribs positioned along the bottom of the flow chamber.

The presently disclosed subject matter also provides methods forassaying biological feature of cultured cells and/or tissues. In someembodiments, the assaying is done in the presence of treatment materialsincluding but not limited to small organic molecules, biochemicals, andthe like. In some embodiments, the assaying is done in the absence oftreatment materials including but not limited to small organicmolecules, biochemicals, and the like. In some embodiments, thepresently disclosed methods comprise (a) growing a cultured cell ortissue on a growth surface present in a flow chamber of the presentlydisclosed subject matter (b) applying a first flow condition and/ortreatment materials to the cultured cell or tissue; and (c) assaying abiological feature of the cultured cell or tissue under the first flowcondition to produce a first analysis of the biological feature of thecultured cell or tissue under the first flow condition with or withouttreatment materials. In some embodiments, the biological featurecomprises a growth rate, an apoptosis or death rate, a morphology,and/or an expression profile of one or more gene products in thecultured cell or tissue before, after, and/or during application of thefirst flow condition. In some embodiments, the assaying comprisesgenerating a gene expression profile of one or more genes in thecultured cell or tissue before, after, and/or during application of thefirst flow condition.

In some embodiments, the presently disclosed methods further compriseapplying a second flow condition with or without treatment materials tothe cultured cell or tissue before and/or after application of the firstflow condition. In some embodiments, the first flow condition and thesecond flow condition are different. In some embodiments, the first flowcondition or the second flow condition comprises a static flowcondition.

In some embodiments, the presently disclosed methods further compriseassaying the biological feature of the cultured cell or tissuesubsequent to and/or while applying the second flow condition to producea second analysis of the biological feature of the cultured cell ortissue under the second flow condition, with or without treatmentmaterials. In some embodiments, the biological feature comprises geneexpression levels of one or more genes in the cultured cell or tissue.In some embodiments, the presently disclosed methods further comprisecomparing the first analysis to the second analysis in order to identifydifferences in a response of the cultured cell or tissue to the firstflow condition as compared to the second flow condition, with or withouttreatment materials. In some embodiments, the biological featurecomprises gene expression levels of one or more genes in the culturedcell or tissue and the comparing step identifies at least one gene forwhich expression differs under the first flow condition as compared tothe second flow condition (alternatively with or without treatmentmaterials) by at least two-fold.

It is thus an object of the presently disclosed subject matter toprovide a flow chamber.

An object of the presently disclosed subject matter having been statedhereinabove, and which is achieved in whole or in part by the presentlydisclosed subject matter, other objects will become evident as thedescription proceeds when taken in connection with the accompanyingFigures and non-limiting examples as best described herein below.

BRIEF DESCRIPTION OF THE FIGURES

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

Exemplary embodiments of the subject matter described herein will now beexplained with reference to the accompanying Figures, wherein likenumerals represent like parts, of which:

FIG. 1 is a perspective view of an exemplary flow chamber 100 of thepresently disclosed subject matter.

FIGS. 2A and 2B are a top view and a bottom view, respectively, ofexemplary inner panel 102 of the presently disclosed subject matter.

FIGS. 3A and 3B are a top view and a bottom view, respectively, ofexemplary outer frame 104 of the presently disclosed subject matter.

FIG. 4 is a cross sectional view along the line 4-4 in FIG. 1 of an isexemplary flow chamber 100 of the presently disclosed subject matter.

FIGS. 5A-5H are perspective views of an exemplary plug 110 of thepresently disclosed subject matter.

FIGS. 6A-6F are perspective views of an exemplary septum 402 of thepresently disclosed subject matter.

FIGS. 7A-7D are schematic sectional views of an exemplary growth surface408 of the presently disclosed subject matter showing exemplarydifferent geometries, obstacles, gap widths, and wall heightsrespectively.

FIG. 8 is a schematic of an exemplary flow chamber of the presentlydisclosed subject matter connected to a flow channel.

FIGS. 9A and 9B are top views of exemplary inner panel 102 of thepresently disclosed subject matter.

FIGS. 10A-10D are computational fluid dynamic model renderings of fluidstreamlines for two viscosities and constant shear stress along apreferred flow channel growth surface.

FIGS. 11A-11F are a series of photomicrographs presenting the results ofa DUOLINK® study examining the association of two proteins, Smad2 andILK, under both static and flow conditions. Human Aortic EndothelialCells were grown to confluence before initiating flow at 1.0 Pa for 20hours, or maintaining static conditions for the same time.

FIG. 12 presents a series of multichannel fluorescence microscopy imagesof cells grown on 1.2 mm thick polystyrene labeled for Akt and F-actinfiber distribution. Magnifications of 20× and 40× are shown.

DETAILED DESCRIPTION

All technical and scientific terms used herein, unless otherwise definedbelow, are intended to have the same meaning as commonly understood byone of ordinary skill in the art. References to techniques employedherein are intended to refer to the techniques as commonly understood inthe art, including variations on those techniques or substitutions ofequivalent techniques that would be apparent to one of skill in the art.While the following terms are believed to be well understood by one ofordinary skill in the art, the following definitions are set forth tofacilitate explanation of the presently disclosed subject matter.

All references listed herein, including but not limited to patents,patent application publications, journal articles, and database entries(e.g., GENBANK® database entries, including all annotations andreferences cited therein) are incorporated herein by reference to theextent that they supplement, explain, provide a background for, or teachmethodology, techniques, and/or compositions employed herein.

Following long-standing patent law convention, the terms “a”, “an”, and“the” mean “one or more” when used in this application, including theclaims. Thus, the phrase “a flow channel” refers to one or more flowchannels, unless the context clearly indicates otherwise.

As used herein, the term “and/or” when used in the context of a list ofentities, refers to the entities being present singly or in combination.Thus, for example, the phrase “A, B, C, and/or D” includes A, B, C, andD individually, but also includes any and all combinations andsubcombinations of A, B, C, and D.

The term “comprising”, which is synonymous with “including”,“containing”, and “characterized by”, is inclusive or open-ended anddoes not exclude additional, unrecited elements and/or method steps.“Comprising” is a term of art that means that the named elements and/orsteps are present, but that other elements and/or steps can be added andstill fall within the scope of the relevant subject matter.

As used herein, the phrase “consisting of” excludes any element, step,and/or ingredient not specifically recited. For example, when the phrase“consists of” appears in a clause of the body of a claim, rather thanimmediately following the preamble, it limits only the element set forthin that clause; other elements are not excluded from the claim as awhole.

As used herein, the phrase “consisting essentially of” limits the scopeof the related disclosure or claim to the specified materials and/orsteps, plus those that do not materially affect the basic and novelcharacteristic(s) of the disclosed and/or claimed subject matter.

With respect to the terms “comprising”, “consisting essentially of”, and“consisting of”, where one of these three terms is used herein, thepresently disclosed and claimed subject matter can include the use ofeither of the other two terms.

The term “about”, as used herein when referring to a measurable valuesuch as an amount of weight, time, dimension, etc., is meant toencompass variations of in some embodiments ±20%, in some embodiments±10%, in some embodiments ±5%, in some embodiments ±1%, and in someembodiments ±0.1% from the specified amount, as such variations areappropriate to perform the disclosed methods and/or to employ thepresently disclosed flow chambers.

Reference will now be made in detail to the description of the presentsubject matter, one or more examples of which are shown in the Figures.Each example is provided to explain the subject matter and not as alimitation. In fact, features illustrated or described as part of oneembodiment can be used in another embodiment to yield still a furtherembodiment. It is intended that the present subject matter cover suchmodifications and variations. Wherever possible, the same referencenumbers will be used throughout the Figures to refer to the same or likeparts. The scaling of the Figures does not represent precise dimensionsof the various elements illustrated therein.

Referring now to the Figures, again wherein like reference numeralsrefer to like parts throughout when possible, a flow chamber inaccordance with one embodiment of the presently disclosed subject matteris referred to generally at 100. Referring in particular to FIGS. 1-4,flow chamber 100 includes inner panel 102 and outer frame 104. In someembodiments, the thickness of inner panel 102 and outer frame 104 is inthe range of about 0.1 to about 1.0 mm, optionally about 0.9 mm. Outerframe 104 includes a skirt 106 that defines the perimeter of flowchamber 100 and also includes a bottom section 302, best seen in FIGS.3B and 4. Inner panel 102 comprises a recess 204 in a lower surface 206thereof and a top view window 114 defined in an upper surface 208thereof. Outer frame 104 includes fluidics holes 116 which are adaptedfor placement for communication with septa holder structures 112 ininner panel 102 when inner panel 102 and outer frame 104 are assembled.Inner panel 102 thus includes septa holders 112, which are adapted toreceive septa 402. Inner panel 102 and outer frame 104 define a flowchannel 404 wherein an inner portion of flow channel 404 is defined byrecess 204 in inner panel 102 and an outer portion of flow channel 404is defined by surface 304 of bottom section 302 of outer frame 104. Insome embodiments, flow channel 404 has dimensions of between about 5 and80 mm long by about 1 and 20 mm wide by about 0.025 and 2.5 mm high. Insome embodiments, the width of flow channel 404 is about 10 mm, itslength is about 60 mm, and its height is about 0.40 mm. Ports 108 areformed in inner panel 102 and permit gas or other exchange with flowchannel 404. Ports 108 can be releasably sealed with plugs 110. In someembodiments, plug 110 can comprise a hydrophobic material, optionally ahydrophobic porous material, a gas permeable material, or other materialas described elsewhere herein. In some embodiments, plug 110 can beself-sealing. In some embodiments, plug 110 can be adapted to fit ontoan end of a standard 1000 μl, 200 μl, or 20 μl pipettor shaft, and/or anautomated liquid handler head or tip. In some embodiments, plug 110 canbe hollow and connected to a filter, optionally a gas filter, which insome embodiments has a porosity of at most 0.2 μm porosity for gasexchange.

Continuing with reference to FIGS. 1-4, top view window 114 and bottomsection 302 can comprise a material through which flow channel 404 canbe observed, such as but not limited to one or more non-birefringentand/or non-auto-fluorescent plastics. In some embodiments, anon-birefringent and/or non-auto-fluorescent plastic is polystyrene. Insome embodiments, bottom section 302 can be thinner at positions overflow channel 404 as compared to other positions. In some embodiments,bottom section 304 can be made from glass and/or contain a section thatcomprises glass. Flow channel 404 includes a growth surface 408, whereincell growth or other activity in flow channel 404 is observed. In someembodiments, surface 304 and growth surface 408 are the same surfacewhen flow chamber 100 is assembled. Inner panel 102 further comprisesflow channel inlet/outlet 202 which provides for communication and isconnection between fluidics holes 116, septum 402 and flow channel 404.In some embodiments, flow channel inlet/outlet 202 acts as a bubbletrap. Further, inner panel 102 comprises a groove 210 adapted to receivewelding rib 306 on outer frame 104 when outer frame 104 and inner panel102 are assembled. When presented as an assembled unit, as shown inFIGS. 1 and 4, flow chamber 100 thus includes inner panel 102 attachedto outer frame 104 via welding rib 306. Welding can be accomplished viaan ultrasonic welding approach or by any other approach that might beapparent to one of ordinary skill in the art upon a review of theinstant disclosure. Before welding occurs, septa 402 are installed insepta holders 112 such that fluidics holes 116 are aligned with thecenter of septa 402. Outer frame 104 seals septa 404 into septa holder112. Septa 402 are adapted to be liquid tight in an assembled flowchamber 100, including when inlet/outlet openings 202 are in fluidcommunication with flow channel 404. Inlet/outlet openings 202 can alsoserve as a bubble trap to capture gas bubbles in entering fluid prior tocontact with the flow channel 404. Further, septa 402 receive fluidicsconnections from a reservoir (not shown in FIGS. 1 through 4) by beingpierced with, for example a needle or other small tube, to introduce orremove flow from flow chamber 100. Indeed, fluid flow can beaccomplished from one fluidic hole 116 as an inlet to an opposed fluidichole 116 that can serve as an outlet. In some embodiments, septa 402create a liquid tight seal around a line used to introduce flow intoflow chamber 100.

Referring now to FIGS. 5A-5H, a plug 110 in accordance with thepresently disclosed subject matter is shown in more detail. Plug 110 cancomprise a flange 502, a post 504, and a stopper 506. Void space 508 isalso defined in the interior of plug 110. Plug 110 is adapted toreleasably seal port 108 particularly via stopper 506. Plug 110 isfurther adapted to retain liquid within flow chamber 100 untilpurposefully removed. In some embodiments, plug 110 can be adapted tofit onto an end of a standard 1000 μl, 200 μl, or 20 μl pipettor shaft,or an automated liquid handler head or tip, via void space 508. In someembodiments, plug 110 can be self-sealing. In other embodiments, plug110 can be porous, or porous and self-sealing. In some embodiments, plug110 can be connected to a filter, optionally a gas filter, which in someembodiments has a porosity of at most 0.2 μm porosity for gas exchange.

Referring now to FIGS. 6A-6F, a septum 402 in accordance with thepresently disclosed subject matter is shown in more detail. Septum 402can comprise a head 602 and a post 604. The center of septum 402 isaligned with fluidics hole 116 and flow channel inlet/outlet 202 toprovide for the flow of fluid into the flow channel 404. Septum 402 iselastomeric, and adapted to create a liquid tight seal to retain liquidwithin flow chamber 100 until purposefully removed.

Referring now to FIGS. 7A-7C, certain features of flow channel 404 aredepicted. Particularly, flow channel 404 can comprise one or more gaps,obstacles, and/or other modifications designed to create one or morevariable fluid dynamic conditions within flow channel 404. As shown inFIG. 7A with a heavy black line, flow channel 404 can include modifiedupper surface 218 and/or lower surface 304 of flow chamber 404.Representative modifications include but are not limited to chemicaland/or physical treatments and/or functionalization by reactive groupsand/or by macromolecules. As best seen in FIG. 7B, flow channel 404 caninclude obstacles 704 that can be of any geometric shape or combinationof shapes, and can be placed in gap 702 between recess 204 in innerpanel 102 and surface 304. Further, as best seen in FIG. 7C, a variablegap 706 between recess 204 in inner panel 102 and surface 304 isprovided so that the height of flow channel 404 can vary, for example,can increase, for at least a portion of its length. As shown in FIG. 7D,the height of the walls of flow channel 404 can vary, for exampleincrease, in a plurality of steps 708.

Referring now to FIG. 8, a flow loop 800 including chamber 100 of thepresently disclosed subject matter is provided. A pump 804 deliversfluid from liquid reservoir 802 via first fluid line 806 to chamber 100via ports 116. Fluid is introduced to flow chamber 100 through septa 402(not shown) and flows through flow channel inlet/outlet 202 through flowchannel 404 (not shown) and out opposite flow channel inlet/outlet 202via septa 402 (not shown) and port 116 to second fluid line 808.Appropriate liquid levels are maintained in reservoir 802 via liquidfeed line 810, and control of other operating parameters can also beincluded, for example system pressure, gas exchange, or pH. Thedirection of the flow is indicated by arrows in FIG. 8, and spent fluidis collected, if desired, for appropriate processing at arrowhead 812.While a representative configuration is provided in FIG. 8, any suitableflow direction or configuration is provided in accordance with thepresently disclosed subject matter as would be apparent to one ofordinary skill in the art upon review of the present disclosure,including but not limited to inclusion of the fluid reservoir within theboundaries of the flow chamber 100.

Referring now to FIGS. 9A and 9B, column position gridlines 902 and rowposition gridlines 904 are superimposed over inner panel 102 of thepresently disclosed subject matter. Gridlines 902 and 904 intersect todefine column/row positions 906 where the wells on a standard 96-wellplate would occur. In some embodiments, each intersection position 906of gridlines 902 and 904 corresponds to the center of a virtual well908. Thus, with respect to top view window 114, approximately fourvirtual wells 908 of a 96-well plate can be encompassed through fourintersection positions 906. Further, ports 108 are located in positions906, and are thus aligned with well positions of a standard 96 wellplate. In accordance with an aspect of the presently disclosed subjectmatter, then, outer frame 104 defines a perimeter of the presentlydisclosed panel chamber 100 that is standardized to facilitate automatedreadout and handling of chamber 100. Accordingly, flow chamber 100 hasoverall dimensions of a standard 6, 12, 24, 48, 96, 384, or 1024 wellmultiwell plate and flow channel 404 is located in a position thatcorresponds to a column/row location of a standard 6, 12, 24, 48, 96,384, or 1024 well multiwell plate. Further, referring back to FIG. 4, asseen by horizontal line 410 across the top of FIG. 4, the height of theouter frame 104 is also a standard height. Thus, again, in accordancewith one aspect of the presently disclosed subject matter the wholedevice layout is designed to facilitate integration with robots, liquidhandlers, and plate readers/high content screening microscopy systems.All of the features of chamber 100 fit into a package that is defined bythe parameters required for automated handling (overall size, height andfeature locations).

As can be seen in FIGS. 1-4 and 9, flow chamber 100 can comprise two ormore flow channels 404. Indeed, flow chamber 100 can comprise in someembodiments two, in some embodiments three, in some embodiments four, insome embodiments five, in some embodiments six, in some embodimentsseven, in some embodiments eight, and in some embodiments up to twelveor more flow channels 404. In such embodiments, each flow channel 404can be individually located in a column/row position that corresponds toa different column/row position of a standard multiwell plate (e.g., astandard 6, 12, 24, 48, 96, 384, or 1024 well multiwell plate). In someembodiments, flow channel 404 is aligned with column/row positions on astandard 96 well plate and/or a standard 384 well plate.

In some embodiments, the inner panel, the outer frame, the one or moreview windows, or any combination thereof are made from one or morenon-birefringent and non-auto-fluorescent plastics. In some embodiments,the inner panel and the outer frame are produced by injection molding.In some embodiments, the overall dimensions of the flow chamber areconsistent with ANSI/SBS well (e.g., plate standards that correspond tostandard 6, 12, 24, 48, 96, 384, and/or 1024 well multiwell plates). Insome embodiments, the flow chamber is provided as a preassembled,presterilized, liquid tight, and tissue culture ready device.

As presented in FIG. 10, an exemplary embodiment of the presentlydisclosed flow chamber generates and maintains parallel fluidstreamlines along surfaces 304/408 within 30 μm of inlet and outlet 202locations. Computational fluid dynamics simulations were performed usingComsol MULTIPHYSICS® Software v. 4.4 for a physiologically relevantarterial shear stress of 1.5 Pa at fluid viscosities or 0.8 (water) and3.0 (blood) cP. The different viscosities result in differential fluidflow rates through the channel to achieve the target shear stress; apeak flow rate of 38.82 ml/min was used for the 0.8 cP case. Theseresults indicate that the fluid dynamics in the channel are stable, andprovide laminar parallel flow over a wide range of operating conditions.FIGS. 10 A and B—streamlines for 0.8 cP fluid at 1.5 Pa. FIGS. 10 C andD—streamlines for 3.0 cP at 1.5 Pa.

The presently disclosed flow chambers can be employed for culturingcells and/or tissues under exposure to fluid flow for the purpose ofgenerating cells or tissues with a desired physiological phenotype thatis related to developmental biology, cardiovascular disease, cancer,inflammation, and/or any other condition that cells and/or tissues froman organism may from time to time experience. Cells of interest can beattached to surface 408 of flow channel 404 and exposed to varioususer-defined fluid flow characteristics with or without treatmentmaterials for a desired length of time.

For example, in some embodiments the presently disclosed flow chamberscan be employed by introducing cells onto growth surface 408, thenreducing or eliminating flow through flow channel 404 to allow for celladhesion to growth surface 408. Once cells are adhered, fluid flow isramped up to a flow rate of interest and held for a desired time period,with or without the introduction of treatment materials. Uponachievement of experimental goals, the induced properties of thecultured cells can be examined at the whole cell, protein, or nucleicacid level.

Cell types that can be tested using the flow chambers and methods of thepresently disclosed subject matter include, but are not limited to,primary mammalian cells (e.g., endothelial cells, epithelial cells,smooth muscle cells, cardiomyocytes, chondrocytes, macrophages, andtransformed cells), stem cells (e.g., embryonic stem cells, adult stemcells, and induced pluripotent stem cells), cell lines (e.g., cancercells, immortalized cell lines, etc.), bacteria, yeast, and any othercell for which examination of growth responses and/or changes inbiological activities under different flow conditions might be desired.In some embodiments, a pure culture of cells is employed, and in someembodiments combinations of different cell types are employed.

Growth surface 408 of flow channel 404 can be modified in various waysto influence the growth and/or attachment of deposited cells.Non-limiting examples of modifications to growth surface 408 includeincluding addition of extracellular matrix components in a molecularlayer or three-dimensional (3D) support (e.g., collagen, fibronectin,laminin, proteoglycans, and/or peptides), molecular layers or 3Dsupports made of other materials (e.g., hydrogels and/or polymers),chemical treatment, and/or other biological materials.

Materials and reagents can be added and removed through flow channelinlet/outlet 202 and/or through port 108. After growth surface 408 isprepared, in some embodiments cells and media can also be added flowchannel inlet/outlet 202 and/or through port 108.

Cells cultured in flow channel 404 and exposed to fluid forces and/orchemical or biochemical treatment materials can be evaluated forbiomarker expression using high-throughput analysis methods. Flowconditions and chemical or biochemical environments that are related toknown characteristics of either healthy or diseased tissues may bechosen for these studies. By way of example and not limitation, expendedculture medium can be removed from flow channel 404 via port 108 and thecells on growth surface 408 washed with appropriate buffer via ports108. A lysis agent can be added to cells on growth surface 408 via port108. After the cells lyse, cellular material can collected through port108 (e.g., by suction via a pipette) and processed for nucleic acidanalysis by microarrays or next-generation sequencing, protein analysisby immunoblot or mass spectroscopy, and/or other methods.

Cells growing on growth surface 408 can also be exposed to a set ofdesired flow conditions in flow channel 404 and then treated withvarious bioactive molecules (e.g., cytokines, chemokines, hormones,growth factors, etc.) and/or other chemical moieties (e.g.,pharmaceutical compounds, contrast agents, organic compounds, inorganiccompounds, etc.) to investigate how physiological responses to thebioactive molecules and/or chemical moieties are affected by variousflow conditions. Cells can also be exposed to a set of desired flowconditions and then treated with molecular biology molecules and/orreagents (e.g., siRNA, shRNA, miRNA, DNA, plasmids, proteins, etc.) todetermine how physiological responses to these molecules are affected byvarious flow conditions.

In some embodiments, cells are treated with biochemicals, chemicalmoieties, and/or molecular biology molecules and/or reagents prior tothe application of flow. In some embodiments, cells are treated withbiochemicals, chemical moieties, and/or molecular biology moleculesand/or reagents after the application of flow. Outcomes are to determinehow the flow conditions affect cell physiology and/or any otherbiologically relevant characteristic of the cells (including, but notlimited to gene expression profiles) and/or how the treatment conditionsinteract with flow conditions to affect cell physiology. In someembodiments, a biologically relevant parameter observed for a celland/or tissue growing in flow chamber 100 prior to the addition of aselected biochemical, chemical moiety, and/or molecular biology moleculeand/or reagent under a given flow condition is compared to the samebiologically relevant parameter observed for a cell and/or tissuegrowing in flow chamber 100 after the addition of a selectedbiochemical, chemical moiety, and/or molecular biology molecule and/orreagent. In some embodiments, a biologically relevant parameter observedfor a cell and/or tissue growing in flow chamber 100 under a first flowcondition is compared to the same biologically relevant parameterobserved for a cell and/or tissue growing in flow chamber 100 under asecond flow condition. In some embodiments, a selected biochemical,chemical moiety, and/or molecular biology molecule and/or reagent isadded to a cell and/or tissue growing in flow chamber 100 before,during, or after the first flow condition is changed to the second flowcondition.

Intact cells can be recovered from growth surface 408 by addingreagents, such as but not limited to, wash buffers, proteases (e.g.,trypsin), or any other reagents that are generally employed to removecells from growth supports or substrates, to flow channel 404 throughport 108 on one end of flow channel 404 and removed through port 108located on the other end of flow channel 404. Recovered cells can beanalyzed by flow cytometry, microscopy, chemiluminescence, microarray,or other assays.

In some embodiments, in situ analysis is performed on fixed or unfixedcells present within flow channel 404. The expression of targetmolecules (e.g., polypeptides, phosphorylated polypeptides, nucleicacids, etc.) can be measured via labeling with appropriate labelingand/or detection reagents that can be applied to the cells. These labelscan bind to the target molecules and provide optical, chemiluminescent,fluorescent, and/or radiological detection of the target molecules.

Single and/or pluralities of flow chamber 100 of the presently disclosedsubject matter can be handled by automated (e.g., robotic moving ofplates and automated liquid processing) or manual mechanisms.

Additionally, cell adhesion experiments can be performed using flowchamber 100 of the presently disclosed subject matter. By way of exampleand not limitation, leukocytes, bacteria, cancer cells, and/or othercells can be flowed over the surface of growth surface 408 and analyzedfor adhesion to growth surface 408, which in some embodiments canalready contain adherent cells and/or other surface modifications.Surface modifications include, but are not limited to addition ofextracellular matrix molecules, ligands, and/or other biological and/orchemical moieties.

Particles, such as nanoparticles or larger entities, can also be flowedover growth surface 408 for determining binding of the nanoparticles orlarger entities to an unmodified or modified growth surface 408 in thepresence or absence of pre-deposited cells. Applications include but arenot limited to cellular toxicity testing, binding kinetics, drugdelivery, and cell targeting testing of nanoparticles, contrast agents,microbubbles, liposomes, and/or other particles.

The flow chambers of the presently disclosed subject matter can beemployed in any method wherein examination of different responses ofbiological molecules, cells, tissues, and/or organs to different flowconditions is desired. By way of example and not limitation, thepresently disclosed flow chambers can be employed for exposingbiological molecules, cells, tissues, and/or organs to a set of desiredflow conditions and then examining the same for flow and/or timedependent differences in relevant biological features and/orphysiological properties.

Alternatively or in addition, biological molecules, cells, tissues,and/or organs can be exposed to a set of desired flow conditions andthen exposed to with particular bioactive molecules (e.g., cytokines,chemokines, hormones, growth factors, etc.) and/or other chemicalmoieties (e.g., pharmaceutical compounds, organic compounds, inorganiccompounds, etc.) to determine how physiological responses to thetreatment conditions are affected by and/or otherwise respond to theflow conditions.

Additionally, biological molecules, cells, tissues, and/or organs can beexposed to a set of desired flow conditions and then treated withmolecular biology molecules and/or reagents (e.g., siRNA, shRNA, miRNA,DNA, plasmids, proteins, etc.) to determine how physiological responsesto the treatment conditions are affected by and/or otherwise respond tothe flow conditions.

Derivatives of the analyses described above are also within the scope ofthe presently disclosed subject matter. For example, treatment withbiochemicals, chemical moieties, and/or molecular biology moleculesand/or reagents can be conducted prior to, during, and/or subsequent tothe application of any particular flow condition, whether testedsingularly or in combination. Potential readouts can include, but arenot limited to determining how any particular treatment conditions(e.g., bioactive molecule exposure) can affect cell physiology incombination with a given flow exposure and/or how the treatmentconditions interact with flow stimulation to affect cell physiology.

Furthermore, the analyses described herein above can also be conductedto compare any desired biological feature under any treatment and/orflow condition of wild type vs. mutant biomolecules, cells, tissues,and/or organs; biological molecules, cells, tissues, and/or organsderived from specific strains and/or genetically modified versions ofany adherent cell type, either prokaryotic or eukaryotic; etc.

Possible endpoints for the methods of the subject matter describedherein can be classified in two groups. In some embodiments, intactcells can be recovered for FACS, flow cytometry, and/or additionalprofiling/cell culture, as well as recovery of cell extracts foranalysis of RNA, DNA, and/or or protein fractions. In some embodiments,intact cells can be recovered by adding appropriate reagents (e.g., washbuffers, trypsin/EDTA, etc.) to the flow channel through one port on topof the channel and removed by the other port. Similarly and in someembodiments, for cell extracts, appropriate wash and/or lysis bufferscan be added through one port and removed through the other. In situanalyses can also be performed on fixed or unfixed cells within the flowchannel. In some embodiments, appropriate buffers and/or reagents canalso be added through one port on top of a flow channel and removed viathe other. In some embodiments, instead of removing the cells or cellextracts, exemplary studies can measure the expression of targetmolecules via labels contained in the reagents applied to the cells.These labels can bind with the target molecules and facilitate opticaland/or radiological detection.

In some embodiments the flow chambers of the presently disclosed subjectmatter can be handled by automated devices (e.g., robotic moving ofplates and automated liquid processing) and/or manually.

An additional assay that can be performed using the flow chambers andmethods of the presently disclosed subject matter is a cell adhesionexperiment. By way of example and not limitation, leukocytes, bacteria,cancer cells, and/or other cells can be flowed over the surface of thepresently disclosed flow device and analyzed for their adhesion to thesurface. In some embodiments, the surface itself can contain adherentcells and/or be a standard and/or modified surface. Surfacemodifications can include, but are not limited to addition ofextracellular matrix molecules, ligands, and/or other biological and/orchemical moieties.

Particle binding studies can also be performed. Particles, including butnot limited to nanoparticles, microparticles, and larger entities, canbe flowed over the flow device surface for determining binding tounmodified and/or modified surfaces in the absence or presence of cells.In some embodiments, cellular toxicity testing, binding kinetics, drugdelivery, and cell targeting testing of nanoparticles, contrast agents,microbubbles, liposomes, and other particles can be performed.

Flow-induced phenotypic alterations can also be tested. Cells and/ortissues can be grown and/or exposed to flow in a flow chamber of thepresently disclosed subject matter for the purpose of generating cellsor tissues with a physiological or pathological phenotype related todevelopmental biology, cardiovascular disease, cancer, inflammation,bones, joints, lymph, lungs or other cells or tissues from an organism.Cells and/or tissues can be mammalian or non-mammalian cells including,but not limited to primary mammalian cells (e.g., endothelial cells,epithelial cells, smooth muscle cells, cardiomyocytes, chondrocytes,macrophages, transformed cells), stem cells (e.g., embryonic stem cells,adult stem cells, induced pluripotent stem cells), cell lines (cancercells, immortalized cell lines, etc.), bacteria, yeast, or other cells.bacterial cells and biofilms, yeast, and/or cells and/or tissues derivedfrom worms, zebrafish, or other organisms. In some embodiments, purecultures or combinations of cell types can be used.

EXAMPLES

The following Examples provide illustrative embodiments. In light of thepresent disclosure and the general level of skill in the art, those ofskill will appreciate that the following Examples are intended to beexemplary only and that numerous changes, modifications, and alterationscan be employed without departing from the scope of the presentlydisclosed subject matter.

Example 1 Biomarker Analysis of Fluid Flow Conditioned Cells

Gene expression differences under two different shear stress conditionswere tested in Human Aortic Endothelial Cells, using a flow chamberdevice.

Table 1 shows the number of genes changed between Human AorticEndothelial Cells exposed to 1.0 Pa wall shear stress for 20 hours ascompared to cells exposed to no flow. Cells were cultured on a collagenI-coated growth surface under static conditions (no flow) untilconfluency was reached. Cells were then either exposed to fluid flow ina flow chamber at 0.2 or 1.0 Pa for 20 hours, or left in static culturefor the same amount of time. At 20 hours, RNA was isolated from thecells using an Ambion MIRVANA™ RNA isolation kit (Life Technologies,Foster City Calif., United States of America) and processed for analysison Affymetrix PRIMEVIEW™ arrays (Affymetrix, Inc., Santa Clara, Calif.,United States of America). Three experiments were performed for eachcondition, providing replicates for microarray analysis.

Table 1 shows the number of genes that were significantly differentbetween pairs of conditions. For cells exposed to 0.2 Pa shear stress,there were 162 genes that significantly changed expression compared tocells not exposed to fluid flow. A similar number of genes were changedfor cells exposed to 1.0 Pa shear stress compared to cells not exposedto flow. However, there were 234 genes changed between cells exposed to0.2 Pa and 1.0 Pa shear stress. These results indicate that both thepresence of flow and the average shear stress magnitude provided eachsignificantly influenced cell physiology. These findings ofdifferentially-expressed genes establish that flow based assays canprovide important information on the physiological state of cells thatis not available from statically conducted experiments. For experimentalwork that targets in vivo physiology, flow assays can provide a morerelevant model than static experiments to study aspects of human oranimal health and disease. The genes identified in the experimentsdescribed herein can be further characterized and tested as targets forhuman therapeutic modulation and/or diagnostics.

TABLE 1 Gene Expression Differences Between Different Shear StressConditions Condition 1 Condition 2 No. of Genes Changed No Shear 0.2 Pa162 1.0 Pa 158 0.2 Pa 1.0 Pa 234

Example 2 Drug Treatment of Fluid Flow Preconditioned and StaticallyCultured Cells

Gene expression differences in Human Aortic Endothelial Cells were alsotested under different shear stress conditions, using a flow chamberdevice, in the presence or absence of the PI3K/Akt and mTOR inhibitorPI-103(3-[4-(4-morpholinyl)pyrido[3′,2′:4,5]furo[3,2-d]pyrimidin-2-yl]-phenol);CAS No. 371935-74-9) at a concentration of 100 nM.

Cells were grown on a collagen-I coated growth surface and treated with100 nM PI-103 in the presence of flow. For the 4 hour time point, cellswere exposed to fluid flow for 16 hours and treated with PI-103 for thelast 4 hours in the presence of flow. Cells were also treated withPI-103 for the entire duration of flow (20 hours). Data was generatedusing RNA extracted as described in Example 1, and analyzed onAffymetrix PRIMEVIEW™ arrays (Affymetrix, Inc.,). Three experiments wereperformed for each condition, providing replicates for microarrayanalysis.

Table 2 shows the number of genes that were significantly differentbetween pairs of conditions. Cells grown only under static conditionsand treated with 4 hours of PI-103 had 130 genes change in comparison tonon-treated cells under static conditions. Cells exposed to PI-103 underflow conditions changed a larger number of genes compared to non-treatedcells under the same flow conditions (see Table 2); drug treatment (4hr) at the low flow condition (0.2 Pa) changed 830 genes while drugtreatment (4 hr) at the high flow condition (1.0 Pa) changed 1563 genes.Treatment of cells with drug for 20 hours resulted in a smaller numberof genes changed for cells exposed to flow (see Table 2). These resultsshow the level of flow exposure (0, 0.2 Pa, or 1.0 Pa) can modifyendothelial gene expression in response to PI-103 treatment, andindicates that cell culture environment is an essential aspect ofexperimental design. PI-103 results were highly divergent betweenstatically cultured cells and both flow conditions, indicating thatstatic culture based assays may be inefficient for predicting howpharmaceutical compounds will interact with living bodies. Byestablishing flow based assays that mimic the flow properties in targettissues, a more relevant physiological environment can be provided forearly stage pharmaceutical experiments.

TABLE 2 Gene Expression Differences Between Different Shear StressConditions and Drug Exposure Times Condition 1 Condition 2^(a) No. ofGenes Changed No Shear  4 hr 130 20 hr 159 0.2 Pa  4 hr 830 20 hr 1131.0 Pa  4 hr 1563 20 hr 119 ^(a)Condition 2 relates to drug exposuretime

Example 3 Identification of Protein Species' Interaction Upon FlowStimulation

Human Aortic Endothelial Cells were cultured on a collagen I-coatedgrowth surface under static conditions (no flow) until confluency wasreached. Cells were then either exposed to fluid flow in a flow chamberdevice at 1.0 Pa for 20 hours, or left in static culture for the sameamount of time. At the 20 hour time point, both sets of cells werewashed well with phosphate buffered saline (PBS), and then fixed for 20minutes at room temperature with 4% p-formaldehyde in PBS. Followingfixation, cells were permeablized with 0.1% TRITON™ X-100 in PBS at roomtemperature for 15 minutes. Once permeablized, cells were well washedagain with PBS and then processed according to the instructions of theDUOLINK® II Kit from Olink Bioscience (Uppsala, Sweden). The DUOLINK®IIKit identifies protein-protein interactions based upon a technique knownas proximity ligation assay. Primary antibodies for the target speciesof Smad2 and Integrin Linked Kinase 1 (ILK) were used at themanufacturer's recommended dilution for immunofluorescence applications.

FIGS. 11A-11E present six (6) confocal laser scanning microscopy panels,three (3) for each condition. FIGS. 11A and 11D show nuclei stained withDAPI, FIGS. 11B and 11E show DUOLINK® signal (indicating protein-proteininteraction), and FIGS. 11C and 11F show the nuclei and DUOLINK® signalmerged. As evident from FIGS. 11A-11F, statically cultured cells showedessentially no interactions between Smad2 and ILK. However, cellsexperiencing 20 hours of flow stimulation at 1 Pa showed significantinteractions between the two proteins.

These experiments demonstrated the ability to employ flow versus staticassays to study cellular based protein activation and interactionphenomena, especially when the targeted interactions might exist invivo. Additionally, the use of microscopy as an assay technique for flowchamber experiments was demonstrated. While not wishing to be bound byany particular theory of operation, it is possible that had theseexperiments been conducted only in statically cultured cells, theinteractions between Smad2 and ILK would likely have gone unobserved.

Example 4

In Situ Detection of Multiple Fluorescently Labeled Proteins byMicroscopy

FIG. 12 presents representative microscopy images of statically grownhuman aortic endothelial cells that were stained for total levels of Aktand F-actin. In this experiment, the ability to detect and distinguishmultiple fluorescent signals through a relatively thick (1.2 mm)polystyrene substrate was investigated. Accomplishment of this imagingtask demonstrated the utility of performing optical assays in flowchambers produced from polystyrene and other plastic materials.Importantly, three fluorophores with distinct excitation and emissioncharacteristics were employed, sequentially imaged on an Olympus FV1000laser scanning confocal microscope, and reconstructed into clearcomposite images. Each individual channel was capable of beingindividually examined for expression characteristics of the targetprotein/structure.

To accomplish this experiment, endothelial cells were cultured in three(3) T-25 flasks until confluent with Lonza EGM-2 growth media (LonzaInc., Allendale, N.J., United States of America) and then rinsed twicewith 5 ml PBS and fixed with 2 ml 4% p-formaldehyde in PBS for 15minutes at room temperature. Following fixation, the cells were rinsedtwice more, and then washed for 5 minutes in 5 ml PBS. A solution of0.1% TRITON™ X-100 in PBS was then added to the cells for 15 minutes atroom temperature to permeablize the cell membrane, and the PBSrinse/wash steps repeated. 5 ml of 5% rabbit serum in PBS was then addedto the cells, and they were allowed to block 6 hours at 4° C. withgentle agitation. A second blocking step using 3% bovine serum albumin(BSA) in PBS was performed for 1 hour at room temperature, and then aprimary rabbit antibody to total Akt (Cell Signaling Technology,Danvers, Mass., United States of America) was added at a dilution of1:500 in a solution of 3% BSA in PBS. Primary antibody was allowed toincubate overnight at 4° C. with gentle agitation. Rinse/wash steps wererepeated with 3% BSA in PBS, and then a secondary antibody conjugatedwith ALEXA FLUOR® 555 (Life Technologies, Foster City, Calif., UnitedStates of America) was incubated with the cells for 1 hour at roomtemperature. Rinse/wash steps were repeated with PBS, and then asolution of Hoechst 33258 (Life Technologies, Foster City, Calif.,United States of America) and FITC-labeled Phalloidin (LifeTechnologies, Foster City, Calif., United States of America) in PBS wereadded at 1 μg/ml each. These components were incubated with the cellsfor 10 minutes at room temperature, and then rinse/wash steps repeated.A fresh 2 ml volume of PBS was added to the cells, and each flask wasimaged on the Olympus FV1000 microscope.

Discussion of the Examples

As set forth herein, the presently disclosed flow chambers and methodscan be employed for assaying a biological feature of cultured cellsand/or tissues, or even isolated biologically interesting moleculesincluding, but not limited to nucleic acids, peptides, polypeptides,polysaccharides, etc. As used herein, the phrase “biological feature”refers to any characteristic of a biomolecule that might be of interestand/or that might be altered by different flow conditions. In someembodiments, a biological feature comprises a growth rate, an apoptosisor death rate, a morphology, and/or an expression profile of one or moregene products in a cultured cell and/or tissue before, after, and/orduring application of one or more different flow conditions.

Additionally, the presently disclosed flow chambers and methods can beemployed in network analysis to analyze, for example, gene expressionand/or protein data under different flow conditions and correlate dataderived therefrom with any other gene expression and/or proteinexpression data from any other source thereby derived to identifybiological pathways that are likely to be involved in the physiologycreated in a given flow chamber experiment and/or flow condition. Thiscan be a powerful technique that can be employed for novel biomarkerdiscovery and/or novel drug target discovery based upon relativelysimple flow experiments employing the presently disclosed flow chambersand/or methods. These data can be acquired in a manner similar to thatdescribed herein above in the EXAMPLES, such as by running microarrayson RNA extracted from flow chamber cultivated/stimulated cells. Suchanalyses can employ specialized software and can be related to geneexpression and/or protein expression and/or phosphorylation data, amongother possible readouts.

Accordingly, the features of the presently disclosed flow chambers arenot available in an existing technology that supports both manual andautomated performance and analysis of flow based cellular assays.

REFERENCES

All references listed below, as well as all references cited in theinstant disclosure, including but not limited to all patents, patentapplications and publications thereof, scientific journal articles, anddatabase entries (e.g., GENBANK® database entries and all annotationsavailable therein) are incorporated herein by reference in theirentireties to the extent that they supplement, explain, provide abackground for, or teach methodology, techniques, and/or compositionsemployed herein.

-   Anderson et al. (2006) The imperative for controlled mechanical    stresses in unraveling cellular mechanisms of mechanotransduction.    BioMed Eng OnLine 5:27.-   Brown & Larson (2001) Improvements to parallel plate flow chambers    to reduce reagent and cellular requirements. BMC Immunol 2:9.-   Buchanan et al. (1999) Relation between non-uniform hemodynamics and    sites of altered permeability and lesion growth at the rabbit    aorto-celiac junction. Atherosclerosis 143:27-40.-   Burns & DePaola (2005) Flow-conditioned HUVECs support clustered    leukocyte adhesion by coexpressing ICAM-1 and E-selectin. Am J    Physiol Heart Circ Physiol 288: H194-H204.-   Chatzizisis et al. (2007) Role of endothelial shear stress in the    natural history of coronary atherosclerosis and vascular remodeling:    molecular, cellular, and vascular behavior. J Am Col Cardiol    49:2379-2393.-   Chiu et al. (2007) Mechanisms of induction of endothelial cell    E-selectin expression by smooth muscle cells and its inhibition by    shear stress. Blood 110:519-528, 2007.-   Dai et al. (2004) Distinct endothelial phenotypes evoked by arterial    waveforms derived from atherosclerosis-susceptible and -resistant    regions of human vasculature. Proc Natl Acad Sci USA.    101:14871-14876, 2004.-   Dekker et al. (2002) Prolonged fluid shear stress induces a distinct    set of endothelial cell genes, most specifically lung Kruppel-like    factor (LKLF2). Blood 100:1689-1698.-   Duan et al. (2010) Shear stress induced changes of membrane    transporter localization and expression in mouse proximal tubule    cells. Proc Natl Acad Sci USA 107:21860-21865.-   Essig & Friedlander (2003) Tubular shear stress and phenotype of    renal proximal tubular cells. J Am Soc Nephrol 14:S33-S35.-   Frangos et al. (1985) Flow effects on prostacyclin production by    cultured human endothelial cells. Science 227:1477-1479, 1985.-   LaMack et al. (2005) Interaction of wall shear stress magnitude and    gradient in the prediction of arterial macromolecular permeability.    Annals Biomed Eng 33:457-464.-   McCann et al. (2005) Non-uniform flow behavior in a parallel plate    flow chamber alters endothelial cell responses. Ann Biomed Eng    33:328-336.-   McKinney et al. (2006) Normal and shear stresses influence the    spatial distribution of intracellular adhesion molecule-1 expression    in human umbilical vein endothelial cells exposed to sudden    expansion flow. J Biomech 39:806-817.-   McNeish (2004) Embryonic stem cells in drug discovery. Nature Rev    Drug Disc 3:70-80.-   Nauman et al. (1999) Quantitative assessment of steady and pulsatile    flow fields in a parallel plate flow chamber. Ann Biomed Eng    27:194-199.-   Rinker et al. (2001) Effect of contact time and force on monocyte    adhesion to vascular endothelium. Biophys J 80:1722-1732.-   Shah et al. (1997) Liver sinusoidal endothelial cells are    responsible for nitric oxide modulation of resistance in the hepatic    sinusoids. J Clin Invest 100:2923-2930.-   Sheikh et al. (2005) Differing mechanisms of leukocyte recruitment    and sensitivity to conditioning by shear stress for endothelial    cells treated with tumour necrosis factor-α or interleukin-1β. Br J    Pharmacol 145:1052-1061.-   Shepherd et al. (2009) Long term shear stress leads to increased    phosphorylation of multiple MAPK species in cultured human aortic    endothelial cells. Biorheology 46:529-538.-   Shepherd et al. (2011) Flow-dependent Smad2 phosphorylation and TGIF    nuclear localization in human aortic endothelial cells. Am J Physiol    Heart Circ Physiol 301:H98-H107.-   Tsai et al. (2007) Laminar flow attenuates interferon-induced    inflammatory responses in endothelial cells. Cardiovasc Res    74:497-505.-   Urbich et al. (2001) Upregulation of TRAF-3 by shear stress blocks    CD40-mediated endothelial activation. J Clin Invest 108:1451-1458.-   Wasserman & Topper (2004) Adaptation of the endothelium to fluid    flow: in vitro analyses of gene expression and in vivo implications.    Vasc Med 9:35-45.-   Yamamoto et al. (2005) Fluid shear stress induces differentiation of    Flk-1-positive embryonic stem cells into vascular endothelial cells    in vitro. Am J Physiol Heart Circ Physiol 288:H1915-H1924.

It will be understood that various details of the presently disclosedsubject matter may be changed without departing from the scope of thepresently disclosed subject matter. Furthermore, the foregoingdescription is for the purpose of illustration only, and not for thepurpose of limitation.

1. A flow chamber comprising: (a) an inner panel having at least oneflow channel formed therein, wherein the at least one flow channel hasan inlet/outlet opening on each end thereof, and further wherein theinlet/outlet openings are adapted to releasably receive a septum; (b)one or more ports adapted for at least liquid communication with the atleast one flow channel to permit liquid or and/or a reagent to be addedthe at least one flow channel, said ports adapted to releasably receivea plug, and optionally wherein the one or more ports are adapted toprovide a liquid-proof seal to the at least one flow channel, andfurther optionally wherein the ports are adapted to be resealable; and(c) an outer frame that defines an outer portion of the at least oneflow channel and that defines a perimeter of the flow chamber. 2.(canceled)
 3. The flow chamber of claim 1, wherein the outer framecomprises a surface upon which cells can be grown in culture. 4.(canceled)
 5. The flow chamber of claim 1, wherein the flow chamber hasoverall dimensions of a standard multiwell plate such as a standard 96well or 384 well multiwell plate, and the at least one flow channel islocated in a position that corresponds to a location on a standardmultiwell plate such as a column location of a standard 96 well or 384well multiwell plate and/or the one or more ports are positioned inlocations aligned with well positions of a standard multiwell plate suchas a standard 96 well or 384 well multiwell plate.
 6. The flow chamberof claim 5, comprising two, three, four, five, six, seven, eight, or upto 12 flow channels, each of which is individually located in a columnposition that corresponds to a different column location of a standardmultiwell plate such as a standard 96 well or 384 well multiwell plate.7. The flow chamber of claim 1, wherein the at least one flow channelhas dimensions of between about 5 and 80 mm long by about 1 and 20 mmwide by about 0.025 and 2.5 mm high.
 8. The flow chamber of claim 1,wherein the at least one flow channel is characterized by one or moregaps, obstacles, and/or other modifications designed to create one ormore variable fluid dynamic conditions within the at least one flowchannel.
 9. The flow chamber of claim 1, wherein the at least one flowchannel has an increasing flow channel height along at least a portionof its length. 10-11. (canceled)
 12. The flow chamber of claim 1,wherein at least an inner surface of the at least one flow channel ischemically and/or physically treated and/or is functionalized byreactive groups and/or by macromolecules. 13-16. (canceled)
 17. The flowchamber of claim 1, wherein at least one of the one or more portscomprises fitted therein a polymer plug, optionally a gas permeableplug. 18-20. (canceled)
 21. The flow chamber of claim 1, wherein theouter frame comprises a skirt defining a perimeter and welding ribspositioned along the bottom of the flow chamber.
 22. The flow chamber ofclaim 21, further comprising one or more viewing windows positionedwithin the perimeter defined by the skirt and between the welding ribs.23-24. (canceled)
 25. The flow chamber of claim 1, wherein the outerframe: (i) is adapted to seal the septum in its correspondinginlet/outlet opening; and (ii) comprises one or more holes to access theseptum for fluidics connections.
 26. The flow chamber of claim 1,wherein the inner panel, the outer frame, or both comprise one or moreview windows through which the at least one flow channel or a cellgrowing thereupon can be observed.
 27. The flow chamber of claim 26,wherein the inner panel, the outer frame, the one or more view windows,or any combination thereof are made from one or more plastics that arenon-birefringent, non-auto-fluorescent, or both. 28-31. (canceled) 32.The flow chamber of claim 1, further comprising at least a first liquidreservoir that is in fluid communication with the at least one flowchannel via a first line attached to the first inlet/outlet opening. 33.A flow chamber comprising: (a) an inner panel having at least one flowchannel formed therein, wherein the at least one flow channel has aninlet/outlet opening on each end thereof, and further wherein theinlet/outlet openings are adapted to releasably receive a septum; (b)one or more ports adapted for at least liquid communication with the atleast one flow channel to permit liquid or and/or a reagent to be addedthe at least one flow channel; and (c) an outer frame that defines anouter portion of the at least one flow channel and that defines aperimeter of the flow chamber; wherein: (i) the outer frame has afootprint equivalent to that of a standard multiwell plate such as astandard 96 well or 384 well multiwell plate; (ii) each of the at leastone flow channels is located in a position that corresponds to a columnlocation of a standard multiwell plate such as a standard 96 well or 384well multiwell plate; and (iii) each of the at least one flow channelscomprises a plurality of virtual wells, each virtual well located in aposition that corresponds to a well location of a standard multiwellplate such as a standard 96 well or 384 well multiwell plate.
 34. Theflow chamber of claim 33, further comprising one or more contact pointsadapted to facilitate interaction of the flow chamber with an automatedplate handling apparatus, a multiwell plate reader, an automatedmicroscopy system or any combination thereof. 35-36. (canceled)
 37. Amethod for producing a flow chamber of claim 21, the method comprisingassembling the inner panel and the outer frame and ultrasonicallywelding the inner panel to the outer frame via the welding ribs.
 38. Amethod for assaying a biological feature of a cultured cell or tissue,the method comprising: (a) growing a cultured cell or tissue on a growthsurface present in the flow chamber of claim 1; (b) applying a firstflow condition to the cultured cell or tissue; and (c) assaying abiological feature of the cultured cell or tissue under the first flowcondition to produce a first analysis of the biological feature of thecultured cell or tissue under the first flow condition.
 39. The methodof claim 38, wherein the biological feature comprises a growth rate, anapoptosis or death rate, a morphology, and/or an expression profile ofone or more gene products in the cultured cell or tissue before, after,and/or during application of the first flow condition. 40-47. (canceled)